Role In Fatty Acid Metabolism Research Articles

Abstract 1415: Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data

Abstract Background Metabolite profiling research offers a deeper insight into biochemical changes in cancer metabolism. Moreover the integrated analysis of transcription, metabolomics and proteomics data can improve the understanding of the underlying biological processes.Material and Methods A set of 254 metabolites was determined by gas chromatography/liquid chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy samples from 95 prostate cancer (PCa) patients. Transcription profiling data obtained from 15 PCa patients by means of Affymetrix U133 arrays was analysed together with public GEO expression data. Expression levels of selected proteins were determined by means of immunohistochemistry and tissue micro array technology in 41 matched frozen tissue samples. The association with clinicopathological variables and clinical outcome was tested. Transcription and metabolomics data were statistically analysed (ANOVA, Mann-Whitney U test) and significant differentially regulated metabolites/genes/proteins were selected.Results Differentially regulated metabolites/genes discrimination between malignant and non-malignant tissues was used for network analysis. Enriched pathways which are involved in PCa progression or recurrence such as carbohydrate and fatty acid metabolism were identified. The role of fatty acid metabolism in PCa was analysed in more detail. Several fatty acids such as cerebronic acid, 2-hydroxybehenic acid, tricosanoic acid showed higher concentrations in malignant than in non-malignant tissues. This finding is in concordance to the observed higher mRNA and protein expression level of fatty acid synthase (FASN) in PCa. In contrast to normal prostate tissue, where protein expression level of FASN was correlated to the level of measured metabolites we found in malignant tissues a deregulation of the corresponding pathway. Conclusion Our integrated analysis of transcription, metabolite and proteomics data confirms and extends the role of several biological pathways which are involved in PCa progression Citation Format: Ulrike Rennefahrt, Hellmuth-A. Meyer, Beate Kamlage, Regina Reszka, Philipp Schatz, Carsten Stephan, Klaus Jung, Dimo Dietrich, Glen Kristiansen. Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1415. doi:10.1158/1538-7445.AM2014-1415

Roles of fatty acid metabolism in the maintenance of self-renewing stem cells

Roles of fatty acid metabolism in the maintenance of self-renewing stem cells

Abstract MP56: Epigenome-wide association study of lipid and inflammatory marker to response fenofibrate in the Genetics of Lipid Lowering Drugs and Diet Network Study

Introduction: There is considerable inter-individual variation in response to lipid-lowering drug therapies. This study aimed to discover the epigenetic factors associated with response to fenofibrate, which may be used to facilitate initiatives in personalized medicine. Methods: We conducted an epigenome-wide association study for lipid and inflammatory marker response to 3 weeks of daily treatment with 160 mg of fenofibrate among 749 participants in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study. For both lipids and inflammatory markers, we analyzed the ratio of the post-3 week lipid or inflammatory marker concentration divided by the baseline concentration. We fit mixed models with methylation status of each CpG as the dependent variable and lipid or inflammatory ratio as the variable of interest adjusting for age, sex, study site, and T-cell purity (fixed effects) and family structure (random effect). A Bonferroni corrected P-value of 1.1x10^-7 was considered significant. Results: Methylation of a CpG site in CPT1A on chromosome 11 was significantly associated with LDL cholesterol response to fenofibrate (P=4.18x10 -8 ). In addition, a CpG site in NEU2 on chromosome 2, was significantly associated with IL2 response (P=7.80x10 -7 ). CPT1A plays an important role in fatty acid metabolism, whereas NEU2 is a type of salidase known to be involved in the development of atherosclerosis. Conclusions: Our findings suggest that methylation of CpG sites within CPT1A and NEU2 are associated with LDL cholesterol and IL2 response to fenofibrate, respectively, and could prove useful for identifying patients who benefit from fenofibrate therapy.

Long Chain Acyl-Coenzyme A Synthetase 4 (BnLACS4) Gene from Brassica napus Enhances the Yeast Lipid Contents

Long-chain acyl-Coenzyme A (CoA) synthetases (LACSs) catalyze the formation of long-chain acyl-CoA, and play important roles in fatty acid metabolism including phospholipids, triacylglycerol (TAG) biosynthesis and fatty acid β-oxidation. Here, we report the characterization of a LACS gene from Brassica napus. It is highly homologous to Arabidopsis LACS4 and thus designated as BnLACS4. The cloned gene BnLACS4 could complement a LACS-deficient yeast strain YB525. It is mainly expressed in flowers and developing seeds where lipid biosynthesis is at high rate in Brassica napus. When transiently expressed in tobacco leaves, BnLACS4 is localized in endoplasmic reticulum (ER), the common site for eukaryotic pathway of lipid biosynthesis. Expression of BnLACS4 in the yeast strain pep4 increased its lipid content. Taken together, our results suggest that BnLACS4 may be involved in lipid biosynthesis in B. napus.

Targeting The Interactions Of Fatty Acid Metabolism With PI3K/mTOR and MAPK As a Novel Therapeutic Strategy In Diffuse Large B-Cell Lymphoma (DLBCL)

BackgroundFatty acid metabolism is altered in several cancers, which includes de novo synthesis by fatty acid synthase (FASN) and increased utilization of fatty acids via β-oxidation to meet cellular energy requirements. PI3K and MEK/ERK signal transduction are primary biological pathways that activate tumor-related fatty acid metabolism. Notably, these concepts have emerged primarily from solid tumor studies; there is a comparative paucity of data in lymphoma. We examined the functional roles of fatty acid metabolism in DLBCL cells and their interaction with oncogenic signal transduction pathways including PI3K, MEK/ERK and hypoxia inducible factor-1 alpha (HIF-1a). We also investigated potential therapeutic implications of targeting fatty acid metabolism for the treatment of DLBCL. MethodsDLBCL cell lines OCI-LY3, OCI-LY19, SUDHL4, and SUDHL10 were examined in this study. Cerulenin (FASN inhibitor), Orlistat (FASN and LPL inhibitor), BEZ-235 (Dual PI3K/mTOR inhibitor), AZD6244 (MEK inhibitor) were used to study the effects of enzyme inhibition on lipid metabolism, cell signaling and cell death. We assessed cell viability with the MTT assay and apoptosis by flow cytometric analysis of Annexin-V/propidium iodide (PI). FASN or LPL mRNAs were quantified in DLBCL cell lines by quantitative RT-PCR as well as through gene expression profiling (GEP) analysis (by cell of origin) using the CaBIG dataset. Further, FASN and associated signaling pathways (MEK, ERK, and HIF-1a) were analyzed by Western blot. Finally, for investigation of potential interactions between FASN and HIF-1a or MAPK signaling, we utilized short hairpin RNA interference (shRNA) to knock out (KO) desired pathways. ResultsGEP analysis showed that genes related to fatty acid synthesis and mTOR were more prominently expressed in germinal center (GC) DLBCL (p=0.0006 vs GC control and p=0.0001 vs non-GC DLBCL), whereas genes involved in utilization of fatty acid for energy, PI3K and MAPK were preferentially expressed in non-GC DLBCL (p<0.0001 vs non-GC control and GC DLBCL). Protein level expression of FASN and CPT1 (relevant to β-oxidation) was readily detectable in all DLBCL cell lines, while the expression of LPL was low, except in SUDHL10 cells. We next examined FASN expression following KO of MEK, ERK, and/or HIF-1a using shRNA in OCI-LY3 and SUDHL10 cells. HIF-1a KO in OCI-LY3 cells and MEK, ERK KO in SUDHL10 significantly decreased FASN expression. Similarly, pharmacological inhibition of PI3K/mTOR or MEK using novel small molecule agents BEZ-235 or AZD6244 (selumetinib), we observed down-regulation of both HIF-1a and FASN expression. Treatment with BEZ-235 under fatty acid deprived conditions resulted in synergistic cell death 60% in OCI-LY3, compared to 25% with BEZ-235 alone. Additionally inhibition of MEK/ERK using AZD6244 or U0126 also resulted in down-regulation of CPT1 relevant to β-oxidation. Pharmacologic treatment with cerulenin, however, resulted in inhibition of MEK and ERK phosphorylation and apoptosis in SUDHL4 and OCI-LY3 cells. Furthermore, treatment with AZD6244 or cerulenin both resulted in time- and dose-dependent cell death (Annexin-V/PI) and decreased proliferation (by MTT) in GC and non-GC DLBCL cell lines (P<0.001). ConclusionsAltogether, we demonstrated that fatty acid metabolism is highly upregulated in DLBCL and that there was preferential gene expression in DLBCL cells in part according to cell of origin. Further, PI3K/mTOR, MAPK and HIF-1a constitutively activated oncogenic pathways in DLBCL appeared here to directly regulate fatty acid metabolism. In addition, we showed that targeting fatty acid metabolism may be harnessed as a potential therapeutic strategy. Continued investigation is warranted to delineate the mechanisms through which MAPK and HIF-1a regulate FASN expression and to determine the in vivo implications of FASN inhibition on DLBCL tumor growth. Disclosures:No relevant conflicts of interest to declare.

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.

Liver Fatty Acid-Binding Protein (L-Fabp) Modifies Intestinal Fatty Acid Composition and Adenoma Formation in ApcMin/+ Mice

Evidence suggests a relationship between dietary fat intake, obesity, and colorectal cancer, implying a role for fatty acid metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid-binding protein (L-Fabp), a dominant intestinal fatty acid-binding protein, regulates intestinal fatty acid trafficking and metabolism, and L-Fabp deletion attenuates diet-induced obesity. Here, we examined whether changes in intestinal fatty acid metabolism following L-Fabp deletion modify adenoma development in Apc(Min)(/+) mice. Compound L-Fabp(-/-)Apc(Min)(/+) mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated, and polyunsaturated fat. L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in adenoma number and total polyp area compared with Apc(Min)(/+)controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp(-/-)Apc(Min)(/+) mice exhibited reductions in cellular proliferation, high-grade dysplasia, and nuclear β-catenin translocation. Intestinal fatty acid content was increased in L-Fabp(-/-)Apc(Min)(/+) mice, and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated fatty acid species with reduced saturated fatty acid species. L-Fabp(-/-)Apc(Min)(/+) mice also showed corresponding changes in mRNA expression of enzymes involved in fatty acid elongation and desaturation. Furthermore, adenomas from L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis, and identify fatty acid trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity, and intestinal tumor formation.

Hepatotoxicity of perfluorooctanoic acid in human hepatocytes using metabonomics

Ultra performance liquid chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry was used to study metabolic profiling of L-02 human liver cell exposed to different doses of perfluorooctanoic acid (PFOA) for 72 h. Principle component analysis method was used for group differentiation and biomarker screening. Dose-dependent distribution of the treated samples with different doses could be observed in scores plots obtained under both positive and negative ionization modes. Furthermore, eighteen metabolites were identified as potential biomarkers which were closely related with the toxicity of PFOA. The identified biomarkers included carnitine and acylcarnitines, nucleosides and nucleoside conjugates, amino acids and amino acid conjugates etc. Among them, significant changes of carnitine and its metabolites were observed in the control group and dose groups, which play an important role in fatty acid metabolism. Meanwhile, some genes involved in cholesterol biosynthesis were upregulated dramatically using gene microarray analysis. The disturbance of cholesterol biosynthesis could have adverse impact on fatty acid metabolism, consequently induce the decrease of carnitine and increase of acylcarnitines in treated groups. Additionally, purine metabolism, tricarboxylic acid cycle, glycosphingolipid metabolism and amino acid metabolism may also be involved in the toxic response to PFOA.

Propionyl-L-carnitine induces eNOS activation and nitric oxide synthesis in endothelial cells via PI3 and Akt kinases

Propionyl-l-carnitine (PLC) is a natural short-chain derivative of l-carnitine (LC), a natural amino acid that plays an important role in fatty acid metabolism. Recent studies suggest that PLC has vascular protective effects. Because of the importance of endothelial nitric oxide synthase (eNOS) and its product, antiatherogenic molecule nitric oxide (NO), in vascular endothelial function, we sought to elucidate that if PLC would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3 Kinase) in cultured human aortic endothelial cells (HAEC). PLC caused eNOS phosphorylation at Ser-1177, and dominant negative Akt and a novel Akt-selective inhibitor MK-2206 inhibited both PLC-mediated phosphorylation and activation of the enzyme. PI3 kinase inhibition also blocked the phosphorylation and activation of eNOS by PLC. Studies with specific drug inhibitors PD173955 and PP2 showed that the non-receptor tyrosine kinase, src, is an upstream stimulator of the PI3 kinase-Akt pathway in this pathway. In addition, PLC significantly decreased intracellular ATP/ADP ratio and activate AMPK, subsequently leading to Src activation. Finally, we demonstrated that the effects of PLC to augment eNOS activity were associated with a net increase in NO release from endothelial cells. NO production following incubation with PLC was abolished in endothelial cells coincubated with L-NAME, PD173955, LY294002, MK-2206 and compound C. In conclusion, PLC, via AMPK/Src-mediated signaling that leads to activation of PI3 kinase and Akt, stimulates eNOS, leading to increased production of NO.

Saturated Very Long Chain Fatty Acids Are Required for the Production of Infectious Human Cytomegalovirus Progeny

Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with13C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.

Ergogenic effect of dietary L-carnitine and fat supplementation against exercise induced physical fatigue in Wistar rats

L-carnitine (LC) plays a central role in fatty acid metabolism and in skeletal muscle bioenergetics. LC supplementation is known to improve physical performance and has become widespread in recent years without any unequivocal support to this practice. A scientific-based knowledge is needed, to understand the implications of LC supplementation on physical fatigue. In current study, we have explored synergistic effects of dietary LC and fat content against physical fatigue in rats. Ninety male Wistar rats were supplemented with different concentrations of LC (0.15, 0.3, and 0.5 %) and fat content (5, 10, and 15 %) through diet in different combinations. Our results elucidated that LC (0.5 %) along with 10 and 15 % fat diet supplemented rats showed significant ergogenic effect. The swimming time until exhaustion was increased by ~2- and ~1.5-fold in rats fed with 10 and 15 % fat diet containing LC (0.5 %). LC supplementation improved the energy charge by increasing the levels of ATP, tissue glycogen, reduced GSH, plasma triglyceride, plasma glucose levels, and enzymatic antioxidant status, i.e., superoxide dismutase, catalase, and glutathione peroxidase. LC supplementation also significantly reduced lipid peroxidation, lactic acid, plasma urea nitrogen, creatinine, creatinekinase, and lactate dehydrogenase levels in various tissues compared to its respective control group. Thus the present study indicates that LC ameliorates the various impairments associated with physical endurance in rats.

High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.

Abstract 1879: Myc induces lipogenesis and suppresses fatty acid oxidation in human P493-6 B-lymphoma cells.

Abstract Myc has been known to control cell growth and proliferation through regulating thousands of genes, of which a substantial amount are involved in metabolism. We have previously shown that Myc increases ribosomal biogenesis, nucleotide metabolism, mitochondrial biogenesis, glycolysis and glutaminolysis in cancer cells. However, Myc's role in fatty acid metabolism is not well-documented, notwithstanding the expectation that Myc induction of cell growth must be accompanied by membrane biogenesis. Other studies have shown that fatty acids can be utilized as energy fuel by cancer cells or as building blocks for proliferation. The P493 human B cell line contains a tetracycline (Tet)-repressible human MYC transgene, permitting robust control of cell growth and proliferation through MYC. Having previously documented that Myc stimulates glucose and glutamine metabolism, we sought to determine whether Myc affects P493 use of fatty acids for oxidation and fatty acid synthesis. We found that resting P493 cells, in which ectopic MYC was off, can use labeled palmitate for respiration, while Myc activated P493 cells demonstrated an inability to oxidize palmitate. We also observe through microarray analysis and RT-PCR that Myc increases the levels of mRNAs for lipogenesis genes. Chromatin immunoprecipitation (ChIP) with an anti-Myc antibody documents that Myc binds to the promoters of lipogenesis genes. Moreover, immunoblot analyses with antibodies available for selected enzymes also indicate that Myc increases the protein levels of ACACA and FASN. Uniformly labeled 13C-glucose and 13C,15N-glutamine were found to be incorporated into lipids when Myc was induced in P493 cells. Cerulenin, an inhibitor of FASN, dramatically inhibits Myc-induced P493 cell proliferation. Similarly, TOFA-inhibition of ACACA suppresses P493 cell proliferation as well. Our data suggest that in P493 Burkitt lymphoma model cells, Myc inhibits fatty acid oxidation and upregulates fatty acid synthesis through transcription of target genes. Citation Format: Arvin Matthew Gouw, Anthony Mancuso, Annie Lee Hsieh, Brian Altman, Michael Wolfgang, Chi Van Dang. Myc induces lipogenesis and suppresses fatty acid oxidation in human P493-6 B-lymphoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1879. doi:10.1158/1538-7445.AM2013-1879

Identification and characterization of Rv0494: a fatty acid-responsive protein of the GntR/FadR family from Mycobacterium tuberculosis

Escherichia coli FadR, a member of the GntR family of transcription factors, plays dual roles in fatty acid metabolism. FadR-DNA binding is inhibited by fatty acyl-CoAs, and thus FadR acts as a sensor of the fatty acid level in bacteria. We have identified FadR-binding sites in the upstream regions of genes showing altered expression after the disruption of fatty acid biosynthesis in Mycobacterium tuberculosis. A FadR homologue in M. tuberculosis, Rv0494, was identified, which binds to its operator in the upstream region of the kas operon. We have shown that FadRMt (Rv0494) directly binds to long-chain fatty acyl-CoA and that binding quenches the intrinsic fluorescence of the purified protein. FadR-DNA binding can be impaired by long-chain fatty acyl-CoA compounds. Overexpression of Rv0494 in Mycobacterium bovis BCG reduced the basal level expression of kas operon genes, thereby suggesting the repressor nature of this protein in fatty acid synthase II regulation. This is the first report, to the best of our knowledge, of a GntR/FadR family protein acting as a fatty acid-responsive transcriptional regulator in M. tuberculosis, suggesting a possible role for this protein in mycolic acid biosynthesis.

Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors.

Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Cultured in Vitro

Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB) during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L) on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL), carnitine palmitoyltransferase IA (CPT IA), long chain acyl-CoA dehydrogenase (ACADL), and acetyl-CoA carboxylase (ACC). Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

The Role of Uncoupling Proteins in Diabetes Mellitus

Uncoupling proteins (UCPs) are anion carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from ATP synthesis. The physiological functions of UCPs have long been debated since the new UCPs (UCP2 to 5) were discovered, and the role of UCPs in the pathogeneses of diabetes mellitus is one of the hottest topics. UCPs are thought to be activated by superoxide and then decrease mitochondrial free radicals generation; this may provide a protective effect on diabetes mellitus that is under the oxidative stress conditions. UCP1 is considered to be a candidate gene for diabetes because of its role in thermogenesis and energy expenditure. UCP2 is expressed in several tissues and acts in the negative regulation of insulin secretion by β-cells and in fatty acid metabolism. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. Several gene polymorphisms of UCP1, UCP2, and UCP3 were reported to be associated with diabetes. The progress in the role of UCP1, UCP2, and UCP3 on diabetes mellitus is summarized in this review.

Molecular Cloning, Characterization, and mRNA Expression of Intestinal Fatty Acid Binding Protein (&lt;i&gt;I-FABP&lt;/i&gt;) in &lt;i&gt;Columba Livia&lt;/i&gt;

Intestinal fatty acid binding protein (I-FABP) is involved in fatty acid transportation in mammals. To verify its role in fatty acid metabolism in birds, the model of pigeon intestinal organ culture was established, and a full-length cDNA of I-FABP was cloned from the intestine of Columba livia by rapid amplification of cDNA ends (RACE) method for the first time. The full length cDNA of C. livia I-FABP was 855 bp, including a 5′untranslated region (UTR) of 34 bp, a 3′UTR of 422 bp and an open reading frame (ORF) of 399 bp encoding a protein of 132 amino acids with the predicted molecular weight of 15.13 kDa. Sequence comparison indicated that I-FABP of C. livia had high identity with other avian I-FABP and belonged to the first subfamily of FABPs. Using quantitative real-time PCR, pigeon I-FABP mRNA in duodenum and jejunum increased progressively with development stage. I-FABP mRNA was expressed at the highest level at 28 day post hatch in the duodenum, while in ileum, it reached the maximum at the day of hatch, and decreased subsequently. The effects of fatty acids on pigeon I-FABP expression were also investigated in vitro. The results showed that significant increases in the pigeon I-FABP mRNA level were induced by linoleic acid and arachidonic acid, whereas I-FABP gene expression appeared to be unaffected by oleic acid and α-linolenic acid. The results indicate that I-FABP may be indicative of intestine development in pigeon, and it could be regulated by n-6 polyunsaturated fatty acids.

Abstract P5-10-04: Metformin mediated upregulation of microRNA-193 triggers apoptosis by decreasing fatty acid synthase

Abstract Background: We have previously shown that the anti-diabetic drug metformin kills triple negative (TN) breast cancer cells lines via apoptosis, at lower IC50 than luminal and HER2 subtypes (presented at ENDO, manuscript in preparation). Metformin reportedly shows greater efficacy against stem cells than differentiated cells, reducing mammosphere formation in in non-adherent culture. Fatty acid synthase (FASN) is an enzyme critical for de novo fatty acid synthesis that is overexpressed in breast cancer. Hypothesis: We hypothesized that miRNAs might be involved in the ability of metformin to preferentially kill TN cell lines at a lower IC50 than luminal A lines. Methods: TN and luminal A (LA) breast cancer cell lines (MDA-468, HCC70 and MCF7, T47D, respectively) were used to study the effects of 10mM metformin action in vitro, at physiological and hyperglycemic culture conditions. Affymetrix chips Human Gene 1.1 and the miRNA 2.0 were utilized to profile gene and miRNA expression at 6 and 24 hrs. Mimics and lentiviral expression vectors were utilized to manipulate miRNA expression and a luciferase reporter was used to confirm miR-193 direct targeting of the FASN 3′ UTR. Results: miR-193 is significantly higher in untreated LA as compared to TN cells. MiR-193 increases 2–4 fold within 6 hrs of metformin treatment in both TN, but not LA cell lines. A predicted target of miR-193, fatty acid synthase (FASN), is decreased by 8 fold following 24 hrs 10mM metformin treatment of the TN breast cancer cells. miR-193 directly targets FASN via a binding site in the 3′ UTR, downregulating gene expression. Restoration of miR-193 to TN lines, causes a dramatic decrease in FASN protein in a dose dependent fashion. Conclusions and future directions: Metformin stimulates an increase in mature miR-193, which mediates the dramatic downregulation of FASN. This occurs coincident with apoptotic cell death. It remains to be determined if FASN is the only relevant direct target of miR-193 in TN cell lines. The addition of exogenous non-targetable FASN, lacking its 3′ UTR, will demonstrate whether this pathway is a primary mechanism of metformin action in TN cells. Future studies will investigate the ability of metformin to inhibit FASN in non-adherent mammosphere (stem cell-like) subpopulations, the role of fatty acid metabolism in mammosphere maintenance and anoikis resistance. Supported by Komen Breast Cancer Foundation Grants KG100575 (ADT, JKR, SME, NSS) and KG090415 (JKR, DRC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-04.

Structure-Guided Discovery of Phenyl-diketo Acids as Potent Inhibitors of M. tuberculosis Malate Synthase

The glyoxylate shunt plays an important role in fatty acid metabolism and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and overexpression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors guided optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics.